Earwood R et al. (2024), The binding of LARP6 and DNAAF6 in biomolecular...

XB-ART-60687

J Biol Chem 2024 Jun 01;3006:107373. doi: 10.1016/j.jbc.2024.107373.

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The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Earwood R, Ninomiya H, Wang H, Shimada IS, Stroud M, Perez D, Uuganbayar U, Yamada C, Akiyama-Miyoshi T, Stefanovic B, Kato Y.

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Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia (PCD). Dynein axonemal assembly factors 6 (DNAAF6), a causative gene of PCD, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multi-ciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were co-localized in biomolecular condensates termed dynein axonemal particles (DynAPs) and synergized to control ciliogenesis. Moreover, Tubulin alpha 1c like (TUBA1CL) mRNA encoding α-Tubulin protein that is a major component of ciliary axoneme was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-Tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-Tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in DynAPs regulates highly expressed α-Tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

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	Figure 1. DNAAF6 interacts with LARP6.A, the domain structure of LARP6. The numbers of amino acid are shown on the FL of LARP6. B, the La motif is crucial for the interaction between LARP6 and DNAAF6. The tagged LARP6 and DNAAF6 proteins were expressed in mammalian cells. HA-tagged full-length LARP6 (FL), LAM, RRM, and C-TER domain of hLARP6 were immunoprecipitated and Flag-tagged hDNAAF6 analyzed in the precipitate. Upper panel: coprecipitated DNAAF6 with LARP6 isoforms; lower panel: proteins in input. C, LARP6 associates with DNAAF6 in an RNA-independent manner. Flag-tagged hDNAAF6 was immunoprecipitated and HA-tagged FL hLARP6 was analyzed in the precipitate. The protein extract was untreated (lanes 1–3) or treated with RNase (lanes 4–6) before coimmunoprecipitation. D, GST-hDNAAF6 pulled down rLARP6. In the middle lane, GST-hDNAAF6 indicated by a white arrowhead and rLARP6 indicated by a black arrowhead were observed. E, interaction of the DNAAF6 mutant proteins with LARP6. HA-tagged FL hLARP6 was immunoprecipitated and WT or mutant DNAAF6 proteins were analyzed in the precipitate (lanes 5–8). Lanes 1 to 4 are proteins in the input. The mutant DNAAF6 D133Y (lane 6, lower panel) could bind to LARP6, while DNAAF6 Q171∗ could not (lane 7, lower panel). C-TER: C-terminal domain; DNAAF, dynein axonemal assembly factor; FL, full length; GST, glutathione-S-transferase; GST-hDNAAF6, GST-fused hDNAAF6; HA, hemagglutinin; hDNAAF6, human DNAAF6; hLARP6, human LARP6; LARP6, La ribonucleoprotein 6; RRM, RNA-recognition motif; rLARP6, recombinant human LARP6.
	Figure 2. DNAAF6 is crucial for ciliogenesis in Xenopus embryos.A, the spatial expression of xDNAAF6 in Xenopus embryos. Developmental stages (St) are indicated on the top of each image. A, animal side; V, vegetal side; a, anterior side; p, posterior side. The scale bars represent 1 mm. B, efficacy of MO knock down of Xenopus DNAAF6. xDNAAF6-2HA mRNA encoding HA-tagged Xenopus DNAAF6 was coinjected with con-MO (40 ng per embryo) or dnaaf6-MO (10 or 20 ng per embryo), and expression of xDNAAF6-2HA protein from stage 20 embryos was examined by Western blot. Loading control: β-actin. C, Xenopus DNAAF6 morphants develop ventral edemas. Left upper panel: uninjected embryos. Right upper panel: edema indicated by white arrows is observed in Xenopus DNAAF6 morphants. Left and right lower panels: formation of edema was rescued by expression of Flag-xDNAAF6 mRNA (WT-RNA) (left lower panel), but not by expression of Flag-hDNAAF6 Q171∗ mRNA (Q171∗-RNA) (right lower panel: edemas are indicated by yellow arrows). The scale bar represents 1 mm. D, scoring of edema formation (number of embryos with edema per total number of examined embryos) in Xenopus DNAAF6 morphants. Numbers of embryos analyzed: con-MO: 65, dnaaf6-MO: 52, dnaaf6-MO+WT-RNA: 53, dnaaf6-MO+Q171∗-RNA: 43. ∗∗∗ p < 0.001. E, dnaaf6-MO inhibited the formation of cilia in Xenopus epidermal MCCs. Cilia were stained by an antibody against acetylated α-tubulin (red), membrane GFP (mGFP) was used as a tracer of injections. Cilia in control MCCs are indicated by white arrowheads, cilia in dnaaf6-MO injected MCCs are indicated by white arrows. The scale bar represents 20 μm. Con-MO, control-MO; DNAAF, dynein axonemal assembly factor; HA, hemagglutinin; hDNAAF6, human DNAAF6; MCC, multiciliated cell; MO, morpholino oligo.
	Figure 3. LARP6 associates and functionally synergizes with DNAAF6 in Xenopus embryos.A, coimmunoprecipitation of xLARP6-2HA with Flag-xDNAAF6, Flag-xDNAAF6 (2-87) or Flag-xDNAAF6 (88-192) (lane 2, 3 or 4 in the right lower panel) using protein extract from stage ten embryos. LARP6 binds the full length of DNAAF6 indicated by a black asterisk, DNAAF6 (2-87) indicated by a black circle and DNAAF6 (88-192) indicated by a white circle. As a note, xDNAAF6 (88-192) includes the protein interacting with HSP90 domain. Expression of xDNAAF6 proteins in the input is shown in the left lower panel. Input or immunoprecipitation of xLARP6 is shown in the right or left upper panel, respectively. B, coimmunoprecipitation of HA-tagged FL xLARP6 with Flag-tagged WT xDNAAF6 indicated by a black circle (lane 2 in the right lower panel) or human DNAAF6 Q171∗ mutant indicated by a white circle (lane 3 in the right lower panel). Expression of the proteins from stage 20 embryos in the input is shown in the left panels (lanes 1–4). C, the colocalization of DNAAF6 and LARP6 in Xenopus epidermal MCCs. Fluorescently labeled xDNAAF6 (GFP-xDNAAF6: green) and xLARP6 (mcherry-xLARP6: red) were expressed in the cytoplasm of MCCs and fluorescence intensity was imaged. The colocalization of GFP-xDNAAF6 and mcherry-xLARP6 was detected as foci. One of these foci is indicated by a white arrow in the cytoplasm of MCCs. Each lower panel shows the view in x and z plane of the z-stack at the yellow line for each upper panel. The scale bar represents 5 μm. D, the localization of xDNAAF6 and cilia in Xenopus epidermal MCCs. GFP-xDNAAF6 (green) was detected as foci in the cytoplasm of MCCs with mRFP-positive cilia indicated by a white arrowhead. A typical focus is indicated by a white arrow. Each lower panel shows the view in x and z plane of the z-stack at the yellow line for each upper panel. The scale bar represents 5 μm. E, the colocalization of xDNAI1 or xDNALI1 and xLARP6 in Xenopus epidermal MCCs. Fluorescently labeled xDNAI1 (GFP-xDNAI1: green) or xDNALI1 (GFP-xDNALI1) and mcherry-xLARP6 were expressed in the cytoplasm of MCCs and fluorescence intensity was imaged. mcherry protein was used as a negative control (a middle panel in the middle row). The colocalization of GFP-xDNAI1 or GFP-xDNALI1 and mcherry-xLARP6 was detected as foci in the cytoplasm of MCCs. A typical focus is indicated by a white arrow. Each lower panel shows the view in x and z plane of the z-stack at the yellow line for each upper panel. The scale bar represents 5 μm. F, the localization of human DNAAF6 and human DNAAF6Q171∗ in stage 23 Xenopus embryo epidermal MCC. Flag-human DNAAF6 WT (hDNAAF6) or Flag-human DNAAF6Q171∗ (hDNAAF6Q171∗) were expressed in Xenopus embryos, and then whole mount immunohistochemistry was performed. Both hDNAAF6 and hDNAAF6Q171∗ were detected as foci indicated by white arrows and yellow arrows in the cytoplasm of epidermal MCCs, respectively. Confocal images of MCCs at an arbitrary z level. The scale bar represents 10 μm. G, LARP6 and DNAAF6 functionally synergize in ciliogenesis of MCCs. mGFP was used as an injection tracer for con-MO (left upper panel) and membrane RFP (mRFP) was used as an injection tracer for dnaaf6-MO and/or larp6-MO (all other panels). Cilia were stained by an acetylated α-tubulin antibody (green). The scale bar represents 20 μm. DNAAF, dynein axonemal assembly factor; HA, hemagglutinin; hDNAAF6, human DNAAF6; LARP6, La ribonucleoprotein 6; MCC, multiciliated cell; MO, morpholino oligo; RFP, red fluorescence protein; xDNAAF6, Xenopus DNAAF6; xDNAI1, Xenopus DNAI; xLARP6, Xenopus LARP6.
	Figure 4. LARP6 showed the high mobility into DynAPs after bleaching.A, time lapse images of mcherry-xLARP6, GFP-xDNAAF6, and GFP-xDANLI1 fluorescence recovery after photobleaching. “s” indicates seconds. Each white arrow indicates a fucus of fluorescently labeled protein. The scale bar represents 10 μm. B, fluorescence recovery after photobleaching kinetics of mcherry-xLARP6 (n = 48), GFP-xDNAAF6 (n = 23), and GFP-xDNALI1 (n = 17). “n” indicates the number of counted DynAPs. The y-axis shows the ratio of fluorescent protein intensity compared with the intensity in the same area before bleaching. DynAP, dynein axonemal particle; LARP6, La ribonucleoprotein 6; xDNAI1, Xenopus DNAI1; xDNAAF6, Xenopus DNAAF6; xLARP6, Xenopus LARP6.
	Figure 5. DNAAF6 is required for fluid flow generated by motile cilia on Xenopus epidermal MCCs.A, the flow generated by cilia in Xenopus epidermal MCCs. Trajectories of beads (purple) were imaged within 0.95 s in MMR buffer of control embryos (left upper panel) and Xenopus DNAAF6 morphants (right upper panel). The trajectories after coinjection of WT-RNA (left lower panel) or Q171∗-RNA (right lower panel) with dnaaf6-MO is shown in the bottom panels. The scale bar represents 1 mm. B, quantification of the speed of beads flow. Number of embryos examined; con-MO: 16, dnaaf6-MO: 15, dnaaf6-MO +WT-RNA: 16, dnaaf6-MO +Q171∗-RNA: 16. ∗∗∗∗ <0.0001. C-TER, C-terminal domain; Con-MO, control-MO; DNAAF, dynein axonemal assembly factor; MCC, multiciliated cell; MMR, Marc's modified Ringer's solution; MO, morpholino oligo; RRM, RNA-recognition motif.
	Figure 6. LARP6 binds xTUBA1CL mRNA and DNAAF6 is important for the expression of α-tubulin proteins in Xenopus epidermal MCCs.A, the strategy of screening for Xenopus LARP6–interacting RNAs in Xenopus embryos is shown. Three independent coimmunoprecipitations were performed and pull-down RNAs were analyzed by RNA-seq. B, peak annotations in the red-highlighted part of chromosome (NC_030729) are shown. Brown bars represent peak annotations. G1-xlLARP6-IP, G2-xlLARP6-IP, and G3-xlLARP6-IP were sample names of coimmunoprecipitation experiments. Numbers in parentheses show the number of peak annotations in a red-highlighted chromosome. C, the spatial expression of xTUBA1CL mRNA in Xenopus embryos. WISH was performed with xTUBA1CL RNA probe. As a note, xTUBA1CL RNA probe likely recognizes mRNAs encoding α-tubulin. St. indicates developmental stages. The scale bar represents 1 mm. D, recombinant human LARP6 protein (rLARP6) binds xTUBA1CL mRNA. Left panel: xTUBA1CL probe (RNA) with the sequence shown was incubated with increasing amounts of rLARP6, and RNA/Protein complexes were resolved on a native acrylamide gel. Right panel: the same experiment with human Collagen type I alpha 2 chain (hCOL1A2) RNA probe was shown as a positive control. Mobility of free RNA and that of RNA/rLARP6 monomer and dimer complexes are shown (arrowheads). The red-highlighted part in xTUBA1CL probe sequence is the precipitated sequence by xLARP6. E, α-tubulin protein is highly expressed in Xenopus epidermal MCCs at the onset of ciliogenesis. The signals of α-tubulin protein (red) at different embryonic stages are indicated by yellow arrowheads (upper panels). The signals of γ-tubulin (green) are indicated by white arrowheads and represent MCCs or precursor MCCs (middle panels). The merged image is shown in the bottom panels. Two panels from the right show different cells at the same stage. The view of x and z plane of the z-stack at the yellow line or the view of y and z plane of the z-stack at the yellow line in left upper large square was shown under or on the right of left upper large square, respectively. The scale bar represents 5 μm. F, the expression of α-tubulin protein was abolished in stage 23 Xenopus DNAAF6 morphants. mRFP mRNA was injected alone into one cell of two-cell stage embryos, and mGFP mRNA was coinjected with con-MO, dnaaf6-MO, dnaaf6-MO/WT-RNA, or dnaaf6-MO/Q171∗-RNA into the other side of cell at four-cell stage embryos. Arrows represent MCCs, into which mGFP mRNA and con-MO, dnaaf6-MO, dnaaf6-MO/WT-RNA, or dnaaf6-MO/171Q∗-RNA were delivered, while arrowheads indicate control MCCs into which only mRFP mRNA was delivered. The expression of α-tubulin protein (cyan) is observed in cilia. The scale bar represents 20 μm. G, the relative mean pixel intensity of α-tubulin protein staining. The relative mean pixel intensity is mean pixel intensity of GFP-positive MCCs relative to mean pixel intensity of adjacent RFP-positive MCCs. ∗: p < 0.05, ∗∗∗∗: p < 0.0001. H, the expression of xTUBA1CL mRNA in stage 20 Xenopus DNAAF6 morphants. SISH was performed with xTUBA1CL RNA probe. Transverse section at the ventral side of embryo is shown. As dnaaf6-MO was coinjected with β-galactosidase mRNA, dnaaf6-MO–injected side is Red-Gal positive side of embryo (left side). The expression level of xTUBA1CL mRNA (purple) is not much different between DNAAF6 knockdown cells and control cells. The area surrounded by a red or black dotted square is enlarged. A red arrow or a block arrow indicates a typical signal of xTUBA1CL mRNA, respectively. The black and white scale bars represent 200 and 50 μm, respectively. Con-MO, control-MO; MCC, multiciliated cell; MO, morpholino oligo; RFP, red fluorescence protein; SISH, section in situ hybridization; xLARP6, Xenopus LARP6; xTUBA1CL, Xenopus tubulin alpha 1c like.

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